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Briefly, purified amplicons were end-repaired, adaptor-ligated, and cleaned up. DNA libraries were prepared using the NEBNext Ultra DNA Library Prep Kit for Illumina, following the manufacturer's recommendations (New England BioLabs, Herts, UK). Purified DNA concentration was determined using a Qubit fluorometer (Life Technologies Corporation, Carlsbad, CA, USA). Amplicons were purified using Expin Gel SV (GeneAll Biotechnology Co., LTD., Seoul, Korea), according to the manufacturer's instructions. These nested-PCR primers amplify a 180-bp fragment of the L1 region. Then, 800 µL of the sample was added to a 1.5 mL tube to wash the pellet, the sample was centrifuged at 8,000 rpm for 5 minutes, and the supernatant was removed.įifty-eight cervicovaginal samples (seven normal, 18 LSIL, 12 HSIL, and 21 cervical cancer samples) previously genotyped by nested-PCR, INNO-LiPA, and electrochemical DNA chip, were selected for HPV genotyping by NGS using the MY/GP primer set for amplification. Approximately 15 mL of each LBC was centrifuged at 3,000 rpm for 5 minutes, and the supernatant was removed. All samples were kept in LBC buffer (ThinPrep Hologic, West Sussex, UK) or phosphate buffered saline (PBS).
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Samples were categorized into four groups based on cytology: normal, low-grade squamous intraepithelial lesions (LSIL), high-grade squamous intraepithelial lesions (HSIL), and cervical cancer.
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All cervical cytology was confirmed by a cytotechnologist and a pathologist. Gynecological samples were selected from the samples obtained during routine Pap smear checkups, investigations, or treatment of patients. All experiments in this research involved conveniently archived samples derived from an earlier study, which examined the prevalence of cervicovaginal HPV infection. The IRB waived the need for consent because the clinical samples were de-identified and anonymous. This study was approved by the Institutional Review Board (IRB) of the Faculty of Medicine, Chulalongkorn University (IRB number 603/2558).